The present invention relates to the field of fermentative production of acylated cephalosporins and their conversion into modified and/or deacylated cephalosporins.
xcex2-Lactam antibiotics constitute the most important group of antibiotic compounds, with a long history of clinical use. Among this group, the prominent ones are the penicillins and cephalosporins. These compounds are naturally produced by the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum, respectively.
As a result of classical strain improvement techniques, the production levels of the antibiotics in Penicillium chrysogenum and Acremonium chrysogenum have increased dramatically over the past decades. With the increasing knowledge of the biosynthetic pathways leading to penicillins and cephalosporins, and the advent of recombinant DNA technology, new tools for the improvement of production strains and for the in vivo derivatization of the compounds have become available.
Most enzymes involved in xcex2-lactam biosynthesis have been identified and their corresponding genes been cloned, as can be found in Ingolia and Queener, Med. Res. Rev. 9 (1489), 245-264 (biosynthesis route and enzymes), and Aharonowitz et al., Ann. Rev. Microbiol. 46 (1992), 461-495 (gene cloning).
The first two steps in the biosynthesis of xcex2-lactam compounds are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-xcex1-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) into the tripeptide LLD-ACV, followed by cyclization of this tripeptide to form isopenicillin N, a penicillin having an a-aminoadipyl side chain. The latter compound contains the typical, xcex2-lactam structure. These first two steps are common in penicillin, cephamycin and cephalosporin producing fungi and bacteria.
In penicillin-producing fungi, like P. chrysogenum, the third step involves the exchange of the hydrophilic xcex1-aminoadipyl side chain of isopenicillin N for a hydrophobic, aromatic side chain by the action of the enzyme acyltransferase. The enzymatic exchange reaction mediated by acyltransferase takes place inside a cellular organelle, the microbody, as has been described in EP448180.
In cephalosporin-producing organisms, the third step is the isomerization of isopenicillin N to penicillin N by an epimerase, whereupon the five-membered ring structure characteristic of penicillins is expanded by the enzyme expandase to the six-membered ring characteristic of cephalosporins.
Currently, there is an increasing need for the fermentative production of xcex2-lactam compounds, especially with regard to the cephalosporin intermediates 7-aminodeacetoxycephalosporanic acid (7-ADCA), 7-aminodeacetylcephalosporanic acid (7-ADAC) and 7-aminocephalosporanic acid (7-ACA). Commercial production of these compounds currently requires extensive chemical synthesis steps, which are expensive and noxious to the environment. Fermentative routes to these compounds are described using recombinant P. chrysogenum strains (EP 532341 and EP 540210).
P. chrysogenum generally is thought to be more suitable than A. chrysogenum for the fermentative production of cephalosporin intermediates, mainly because the xcex2-lactam biosynthetic capacity of P. chrysogenum is higher than of A. chrysogenum, due to extensive strain improvement. For the cephalosporin C producer A. chrysogenum, strain improvement has started much later than for P. chrysogenum, and additionally no amplification of cephalosporin biosynthetic genes was observed (Smith et al. Curr. Genet. 19 (1991), 235-237), contrary to the penicillin biosynthetic genes which are amplified indeed (Smith et al. Mol. Gen. Genet. 216 (1 989), 492-497; Barredo et al. Curr. Genet. 16 (1989), 453-459; Fierro et al., Proc. Natl. Acad. So;. 92 (1995), 6200-6204).
Recently, it was observed that desacetoxy-cephalosporin was formed in a P. chrysogenum strain expressing expandase, implicating that P. chrysogenum might contain an epimerase activity as well (Alvi et al., J. Antibiotics 48 (1995), 338-340). This phenomenon diminishes the supposed advantage of P. chrysogenum above A. chrysogenum as a production organism for the fermentative production of extractable cephalosporins.
A chrysogenum strains which express the Penicillium acyltransferase gene have been described (European Patent EP 357119, Gutixc3xa9rrez et al., Mol. Gen. Genet. 225 (1991), 56-64), but only the production of penicillin G with said recombinant Acremonium strains is disclosed.
Crawford et al. (Bio/Technology 13 (1995), 58-62) suggest the production of adipyl-cephalosporins by feeding adipic acid to an acyltransferase expressing Acremonium strain. However, it is also indicated that this approach would result in a mixture of cephalosporins, with adipyl as well as aminoadipyl side chains, which would present difficulties for downstream processing.
The present invention discloses a fermentative process for the production of an N-acylated cephalosporin derivative.
Specifically, the process of the invention comprises the fermentation of an Acremonium strain in the presence of a suitable acyl side chain precursor, wherein said Acremonium strain is transformed with an expression cassette comprising an acyltransferase coding sequence and transformants are selected wherein said acyltransferase coding sequence is expressed to a level which leads to a production level of said N-acylated cephalosporin derivative which is similar to or higher than the production level of xcex1-aminoadipyl-7-cephalosporin derivatives.
The process of the invention further comprises the fermentation of an acyltransferase-expressing Acremonium strain in the presence of a suitable acyl side chain precursor, wherein said strain does not express hydroxylase and/or acetyltransferase activity.
The resulting N-acylated cephalosporin derivative is subsequently recovered from the culture fluid.
The N-acylated cephalosporin derivative produced according to the invention is used for the preparation of semisynthetic cephalosporins. Alternatively, the N-acylated cephalosporin derivative is deacylated by chemical or enzymatical means, to produce 7-ADCA, 7-ADAC or 7-ACA.
The present invention discloses a fermentative process for the production of N-acylated cephalosporin derivatives, with the proviso that said N-attached acyl group is not the naturally occurring xcex1-aminoadipyl group. Specifically, the present invention discloses a process for the production of N-acylated cephalosporin derivatives using Acremonium as the production organism, wherein Acremonium expresses acyltransferase activity and is cultured in the presence of a suitable N-acyl side chain precursor.
A xe2x80x9csuitablexe2x80x9d N-acyl side chain precursor is understood to be a side chain precursor which is acceptable to the enzyme acyltransferase as well as to be a side chain precursor which produces a penicillin derivative which is amenable to ring expansion by the enzyme expandase. The xcex1-aminoadipyl side chain present in natural penicillin N-derived cephalosporin compounds is understood not to be covered by the term xe2x80x9csuitablexe2x80x9d N-acyl side chain.
Examples of such suitable acyl side chain precursors are adipic acid, thiodipropionic acid and carboxymethylthiopropionic acid.
In the process of the invention, an Acremonium strain is used which expresses an acyltransferase gene to a high level. A high expression level is important to minimize the production of xcex1-aminoadipyl-cephalosporin derivative relative to the production of N-acyl-cephalosporin derivative.
To obtain Acremonium strains with a high production level of N-acyl-cephalosporin derivatives relative to the level of xcex1-aminoadipyl-cephalosporins, said strains should have a sufficiently high acyltransferase expression level. A sufficiently high acyltransferase expression level is obtained by using for instance a strong promoter to direct expression of the enzyme. A sufficiently high acyltransferase expression level is further obtained by selecting fungal strains containing multiple copies of an acyltransferase expression cassette.
The Acremonium strain used in the process of the present invention has an acyltransferase expression level which results in a production level of an N-acyl-cephalosporin derivative which is higher than the production level of the xcex1-aminoadipyl-cephalosporin. Preferably, the production level of the N-acyl-cephalosporin derivative is 1.5-3 times higher, more preferably 3-10 times higher, most preferably more than 10 times higher than the production level of the xcex1-aminoadipyl-cephalosporin.
In the process of the invention, the acyltransferase-expressing Acremonium strain is fermented according to common technology. During fermentation, a suitable acyl side chain precursor is fed to the fungal cells. The present invention shows that the acyltransferase ensures the exchange of the xcex1-aminoadipyl side chain in isopenicillin N for said acyl side chain which is fed during fermentation. The resulting acyl-6-APA derivative is subsequently expanded to the acyl-7-ADCA derivative. Depending on the presence of additional enzymes of the cephalosporin biosynthesis pathway, acyl-7-ADCA may be further converted to acyl-7-ADAC or acyl-7-ACA.
In the process of the invention, it is an option to additionally overexpress the gene encoding the enzyme responsible for activation of the side chain precursor, i.e. the appropriate acyl-coA ligase. When using adipic acid as the side chain precursor, said ligase is adipyl-coA ligase.
The use of an Acremonium strain for the fermentative production of N-acylated cephalosporin derivatives has several advantages above the use of a Penicillium strain. Since Acremonium naturally produces cephalosporins, the organism is better equipped than Penicillium to secrete cephalosporin compounds. In addition, xcex2-lactam biosynthetic enzymes from Acremonium may have a longer halflife than those from Penicillium, a difference in stability which is most probably due to differences in the level of endogenous proteolytic enzymes between the two fungi. Moreover, Acremonium strains already possess a reasonable xcex2-lactam production level while the xcex2-lactam biosynthetic genes are still present as single copies. This implicates that amplification of these genes in Acremonium potentially may lead to a substantially higher xcex2-lactam production level.
The use of an acyltransferase-expressing Acremonium strain for the fermentative production of N-acylated cephalosporin derivatives typically results in the formation of an acyl-7-ACA compound. In that regard, a further advantage of Acremonium above Penicillium is applicable. For acyl-7-ACA production with Acremonium, only one enzyme, acyltransferase, needs to be overexpressed, implying that it is relatively easy to obtain a strain with a high expression level of said enzyme, in contrast, for the fermentative production of N-acylated 7-ACA derivatives using P. chrysogenum, three different enzyme activities have to be recombinantly expressed in this organism, i.e. an expandase, a hydroxylase, and an acetyltransferase.
To allow for the production of other N-acylated cephalosporin derivatives using Acremonium, such as acyl-7-ADCA or acyl-7-ADAC, a further genetic engineering of an Acremonium strain is necessary. Specifically, for the production of acyl-7-ADCA or acyl-7-ADAC the gene encoding hydroxylase or acetyltransferase, respectively, is inactivated, in addition to introduction of a gene encoding acyltransferase.
In one aspect of the invention, an Acremonium strain expressing an acyltransferase enzyme is prepared by transformation of an Acremonium strain with an expression cassette comprising an acyltransferase coding sequence, wherein said coding sequence may be present as a cDNA or as a genomic DNA fragment. Preferably, the acyltransferase gene originates from Penicillium (the penDE gene) or from A. nidulans. 
The acyltransferase expression cassette comprises an acyltransferase coding sequence provided with its native 5xe2x80x2 and 3xe2x80x2 regulatory sequences (transcription and/or translation initiation and termination sequences or with 5xe2x80x2 and 3xe2x80x2 regulatory sequences originating from a gene other than acyltransferase. Preferably, the acyltransferase coding sequence is provided with 5xe2x80x2 and 3xe2x80x2 regulatory sequences which give rise to a high level of transcript and corresponding protein.
Examples of suitable 5xe2x80x2 and 3xe2x80x2 regulatory sequences, i.e. promoters and terminators, providing for recombinant gene expression in filamentous fungus host cells are mentioned in Van den Hondel et al. (in: More Gene Manipulations in Fungi, Eds. Bennett and Lasure (1991), 396-427). Examples of strong promoters functionable in Acremonium are the A. chrysogenum isopenicillin N synthase promoter (Skatrud et al. Curr. Genet. 12 (1987), 337-348, Kuck et al. Appl. Microbial. Biotechnol. 31 (1989), 358-365) or the A. nidulans glyceraldenyde-3-phosphate promoter (Smith et al. Gene 114 (992), 211-216). Transcriptional terminators can be obtained from the same genes as well.
In a further aspect of the Invention, an acyltransferase-expressing Acremonium strain is used as a host for further transformations, to allow for the production of other N-acylated cephalosporins than acyl-7-ACA.
To obtain an Acremonium strain producing an acyl-7-ADAC, it is necessary to eliminate cephalosporin C acetyltransferase activity in said strain. This is done by inactivation of the gene encoding cephalosporin C acetyltransferase (cefG) in an Acremonium strain, in addition to providing said strain with an acyltransferase expression cassette.
In a similar way, to obtain a strain producing an acyl-7-ADCA, the gene encoding hydroxylase (cefEEF) is inactivated in an Acremonium strain, in addition to providing said strain with an acyltransferase expression cassette. Since in Acremonium hydroxylase and expandase are two activities of the same enzyme molecule, inactivation of the hydroxylase gene simultaneously eliminates expandase activity. Therefore, a gene encoding an expandase with only minor hydroxylase activity, such as the cefE gene from Streptomyces clavuligerus (Kovacevic et al. J. Bact. 171 (1989), 754-760) or from Nocardia lactamdurans, is simultaneously or subsequently introduced in said Acremonium strain.
Typically, inactivation of a gene is performed by disrupting the coding sequence of the gene to be inactivated.
Inactivation of the cefG or cefEF gene is done using the known cefG gene sequence (Mathison et al. Curr. Genet. 23 (1993), 33-41: Gutierrez et al. J. Bact. 174 (1992), 3056-3064) or cereF gene sequence (Samson et al. Bio/Technology 5 (1987), 1207-1214) and procedures as described by Hoskins et al. (Curr. Genet. 18 (1990), 523-530), Karhunen et 21. (Mol. Gen. Genet. 241 (1993), 515-522) and Suominen et al. (Mol. Gen. Genet. 241 (1993), 523-530).
Briefly, an Acremonium strain is transformed with a so-called disruption cassette. Said disruption cassette comprises a detectable DNA fragment, provided with 5xe2x80x2 and 3xe2x80x2 flanking sequences which are homologous to the target gene to be inactivated, i.e. the cefG or cefEF gene. Said flanking sequences must be of sufficient length to allow homologous recombination into the target gene. For this purpose, the flanking sequences should comprise at least 1 kb, preferably at least 2 kb and more preferably at least 3 kb target gene sequences. The detectable DNA fragment integrates in the target gene by homologous recombination, either by a single cross-over recombination event, resulting in an insertion of the detectable DNA fragment in the target gene, or by a double cross-over recombination event, resulting in a replacement of target gene sequences by the detectable DNA fragment (in: More Gene Manipulations in Fungi, Eds. Bennett and Lasure (1991), 51-79).
The detectable DNA fragment is a DNA fragment of which the correct integration in the DNA of an Acremonium transformant, i.e. integration in the target gene to be inactivated, is conveniently detectable, e.g. by Polymerase Chain Reaction (PCR) technology and/or Southern hybridisation.
In one embodiment of the invention, the detectable DNA fragment comprises an expression cassette providing for expression of a selection marker.
In another embodiment of the invention, disruption of the cefEF and expression of the cefE gene is achieved in a single transformation event, using a disruption/expression cassette for transformation. Said disruption/expression cassette comprises a disruption cassette directed to the cefEF gene, said cefEF disruption cassette comprising a detectable DNA fragment which is an expression cassette providing for expression of The cefE gene. Next to correct integration of the disruption expression cassette, transformed Acremonium strains are analyzed for expandase expression by checking the nature of the formed N-acyl-cephalosporin derivative.
The desired expression, disruption or disruption/expression cassette is transformed to a suitable Acremonium host strain. Procedures for transformation of A. chrysogenum are well known in the art (Queener et al. Microbiology, Am. Soc. for Microbiology (1985), 468-472; Skatruo et al. Curr. Genet. 12 (1987), 337-348; Whitehead et al. Gene 90 (1990), 193-198). To select transformed cells from the nontransformed background, a suitable selection marker is used. Selection markers to be used for selection of fungal transformants are well Known in the art, The selection marker can reside on the same DNA fragment as the desired construct or, alternatively, can reside on a different DNA fragment or vector.
Transformants obtained after the selection procedure are subsequently analyzed for the presence of the desired characteristic, i.e.:
a suitably high production level of the acyl-7-ACA derivative relative to the level of xcex1-aminoadipylcephalosporin compounds. HPLC is a suitable method to discriminate between acyl-7-ACA and xcex1-aminoadipyl-cephalosporin compounds,
a correct integration of the disruption or disruption/expression cassette using Southern hybridisation and/or PCR technology,
analysis of the nature of the produced N-acylated cephalosporins using HPLC, to check the inactivation of the acetyltransferase and/or hydroxylase gene and/or expression of the re-introduced expandase gene.
The exact order in which the various transformations are performed is not critical to the invention. In a preferred embodiment, an Acremonium strain firstly is transformed with an acyltransferase expression cassette, whereupon a strain with a suitable acyltransferase expression level is selected and used as a host for further transformations.
The present invention further discloses a process for the recovery of an N-acylated cephalosporin derivative, e.g. an acyl-7-ACA derivative, e.g. adipyl-7-ACA, from the fermentation broth of an acyltransferase-expressing Acremonium strain using specific solvents. In this recovery process, the N-acylated cephalosporin derivative is isolated with preference relative to contaminating cephalosporin intermediates having xcex1-aminoadipyl side chains. This recovery process is particularly useful if the level of contaminating xcex1-aminoadipyl cephalosporin derivatives in the fermentation broth is relatively high, e.g. similar to the N-acylated cephalosporin level.
Specifically, adipyl-7-ACA is recovered from the fermentation broth by extracting the broth filtrate with an organic solvent immiscibie with water at a pH of lower than about 4.5 and back-extracting the same with water at a pH between 4 and 10.
The broth is filtered and an organic solvent immiscible with water is added to the filtrate. The pH is adjusted in order to extract adipyl-7-ACA from the aqueous layer. The pH range has to be lower than 4.5; preferably between 4 and 1, more preferably between 2 and 1. In this way, adipyl-7-ACA is separated from many other impurities present in the fermentation broth, especially from the aminoadipyl cephalosporin intermediates. Preferably a smaller volume of organic solvent is used, e.g. half the volume of solvent relative to the volume of aqueous layer, giving a concentrated solution of adipyl-7-ACA, so achieving reduction of the volumetric flow rates. A second possibility is whole broth extraction at a pH of 4 or lower. Preferably the broth is extracted between pH 4 and 1 with an organic solvent immiscible with water.
Any solvent that does not interfere with the cephalosporin molecule can be used. Suitable solvents are, for instance, butyl acetate, ethyl acetate, methyl isobutyl ketone, alcohols like butanol etc. Preferably 1-butanol or isobutanol are used.
Hereafter adipyl-7-ACA is back extracted with water at a pH between 4 and 10, preferably between 6 and 9. Again the final volume can be reduced. The recovery can be carried out at temperatures between 0 and 50xc2x0 C., and preferably at ambient temperatures.
The above recovery process is also suitable for the preparation of thiodipropionyl- and carboxymethyl-thiopropionyl-7-ACA derivatives, and for the preparation of other N-acylated cephalosporins, like adipyl-, thiodipropionyl-and carboxymethyl-thiopropionyl-7-ADCA derivatives.
The N-acylated cephalosporin derivatives produced by the process of the invention are conveniently used as an intermediate for the chemical synthesis of semisynthetic cephalosporins, since the 7-aminogroup is adequately protected by presence of an appropriate acyl side chain.
Alternatively, the N-acylated cephalosporin derivatives are deacylated in a one-step enzymatical process, using a suitable enzyme, e.g. Pseudomonas SE83 acylase.
Preferably, an immobilized enzyme is used, in order to be able to use the enzyme repeatedly. The methodology for the preparation of such particles and the immobilization of the enzymes have been described extensively in EP 222462. The pH of the aqueous solution has a value of, for example pH 4 to pH 9, at which the degradation reaction of cephalosporin is minimized and the desired conversion with the enzyme is optimized. Thus, the enzyme is added to the aqueous cephalosporin solution while maintaining the pH at the appropriate level by, for instance, adding an inorganic base, such as a potassium hydroxide solution, or applying a cation exchange resin. When the reaction is completed the immobilized enzyme is removed by filtration. Another possibility is the application of the immobilized enzyme in a fixed or fluidized bed column, or using the enzyme in solution and removing the products by membrane filtration. Subsequently, the pH of the aqueous layer is adjusted to 2 to 5. The crystalline deacylated cephalosporin is then filtered off.
The deacylation can also be carried out chemically as known in the prior art, for instance via the formation of an iminochloride side chain, by adding phosphorus pentachloride at a temperature of lower than 10xc2x0 C. and subsequently isobutanol at ambient temperatures or lower.
The recovery process of the present invention also envisages the option to prepare a deacylated cephalosporin from a mixture of xcex1-aminoadipyl- and an N-acylated cephalosporin derivative, using one and the same process. Both derivatives are isolated from the broth by a column step as described by Tischer et al. (in: Enzyme Engineering XI, Eds. Clark and Estell (1992), 502-509). The mixture of xcex1-aminoadipyl- and N-acylated cephalosporin derivatives is then subjected to a D-amino acid oxidase treatment to convert the xcex1-aminoadipyl-7-cephalosporin derivative to the corresponding glutaryl derivative. The glutaryl as well as the acyl side chains are subsequently removed from the cephalosporin backbone using for instance Pseudomonas SE83 acylase.